The synthesis or replication of DNA molecule can be divided into three stages
- Initiation (Formation of Replisome)
- Elongation (Initiation of synthesis and elongation)
The replication begins at a specific initiation point called OriC point or replicon. (Replicon: It is a unit of the genome in which DNA is replicated; it contains an origin for initiation of replication) It is the point of DNA open up and form open complex leading to the formation of prepriming complex to initiate replication process.
The OriC site is situated at 74″ minute near the ilv gene. The OriC site consists of 245 basepairs, of which three of 13 basepair sequence are highly conserved in many bacteria and forms the consensus sequences (GATCTNTTNTTTT). Close to OriC site, there are four of 9 basepair sequences each (TTATCCACA).
The sequence of reactions in the initiation process is as follows:
a) Dna A protein recognizes and binds up to four 9bp repeats in OriC to form a complex of negatively supercoiled OriC DNA wrapped around a central core of Dna A protein monomers. This process requires the presence of the histone like HU or 1 HC proteins to facility DNA bending.
b) Dna A protein subunits then successively melt three tandemly repeated 13bp segments in the presence of ATP at >=22*C (open complex).
c) The Dna A protein then guides a Dna B – Dna C complex into the melted region to form a so called prepriming complex. The Dna C is subsequently released. Dna B further unwinds open complex to form prepriming complex.
d) DNA gyrase, single stranded binding protein (SSB), Rep protein and Helicase – II are bound to prepriming complex and now complex is called as priming complex.
e) In the presence of gyrase and SSB, helicases further unwinds the DNA in both directions so as to permit entry of primase and RNA polymerase. Then RNA polymerase forms primer for leading strand synthesis while primase in the form of primosome synthesis primer for lagging strand synthesis.
f) To the above complex, DNA polymerase – III will bind and forms replisome.
REPLISOME: It is the multiprotein structure that assembles at the bacterial replicating fork to undertake synthesis of DNA. It contains DNA polymerase and other enzymes.
Now the stage is set for the initiation of synthesis and the elongation to proceed. But this occurs in two mechanistically different pathways in the 5′–>3′ template strand and 3′–>5′ template strand.
a) Initiation of synthesis and Elongation on the 5′–>3′ template (synthesis of leading strand) (If replication fork moves in 3′–>5′ direction)
The DNA daughter strand that is synthesized continuously on 5′–>3′ template is called leading strand. DNA pol-III synthesizes DNA by adding 5′-P of deoxynucleotide to 3′-OH group of the already presenting fragment. Thus chain grows in 5′–>3′ direction. The reaction catalyzed by DNA pol-III is very fast. The enzyme is much more active than DNA pol – I and can add 9000 nucleotides per minute at 37*C. The RNA primer that was initially added by RNA polymerase is degraded by RNase.
b) Initiation of synthesis and Elongation on 3′–>5′ template when fork moves in 3′–>5′ direction (Synthesis of lagging strand)
The daughter DNA strand which is synthesized in discontinuous complex fashion on the 3′–>5′ template is called lagging strand. It occurs in the following steps:
i) Synthesis of Okazaki fragment:
To the RNA primer synthesized by primosome, 1000-2000 nucleotides are added by DNA pol-III to synthesis Okazaki fragments.
ii) Excision of RNA primer:
When the Okazaki fragment synthesis was completed up to RNA primer, then RNA primer was removed by DNA pol – I using its 5′–>3′ exonuclease activity.
iii) Filling the gap (Nick translation)
The gap created by the removal of primer, is filled up by DNA pol – I using the 3′-OH of nearby Okazaki fragment by its polymerizing activity.
iv) Joining of Okazaki fragment: (Nick sealing)
Finally, the nick existing between the fragments are sealed by DNA ligase which catalyze the formation of phosphodiester bond between a 3′-OH at the end of one strand and a 5′ – phosphate at the other end of another fragment. The enzyme requires NAD for during this reaction.
Termination occurs when the two replicating forks meet each other on the opposite side of circular E.Coli DNA. Termination sites like A, B, C, D, E and F are found to present in DNA. Of these sites, Ter A terminates the counter clockwise moving fork while ter C terminates the clockwise moving forks. The other sites are backup sites. Termination at these sites are possible because, at these sites tus protein (Termination utilizing substance) will bound to Dna B protein and inhibits its helicase activity. And Dna B protein released and termination result.
After the complete synthesis, two duplex DNA are found to be catenated (knotted). This catenation removed by the action of topoisomerase. Finally, from single parental duplex DNA, two progeny duplex DNA synthesized.
REGULATION OF PROKARYOTIC REPLICATION:
Especially initiation of replication is regulated. Dna A protein when available in high concentration then ratio of DNA to cell mass is quiet high but at low Dna A concentration, the ratio found to be low. This shows that Dna A protein regulates the initiation of replication.
The sequence most commonly methylated in E.Coli is GATC including in three of 13mer sequence. Thus, the observation that E.Coli defective in the GATC methylation enzyme are very inefficiently replicated, suggests that the DNA replication trigger also responds to the level of OriC methylation.